CN112697561A - Sample pretreatment method for determining content of sodium hyaluronate in electrospinning membrane material - Google Patents
Sample pretreatment method for determining content of sodium hyaluronate in electrospinning membrane material Download PDFInfo
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 4
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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Abstract
The invention provides a sample pretreatment method for determining the content of sodium hyaluronate in an electrospinning membrane material, which comprises the following steps: and (3) ultrasonically leaching the electrospinning membrane material containing sodium hyaluronate by using water as a leaching medium, collecting the leaching solution, and measuring the content of the sodium hyaluronate in the leaching solution. The invention provides a sample pretreatment method for simply, conveniently, quickly and efficiently leaching hydrophilic substances such as sodium hyaluronate from an electrospun polymer and then measuring the hydrophilic substances; the sodium hyaluronate under the leaching condition has high recovery rate and good leaching effect.
Description
Technical Field
The invention relates to a sample pretreatment method for measuring the content of sodium hyaluronate in an electrospinning membrane material, in particular to the detection of active ingredients introduced by various polymer materials prepared by electrospinning technology, such as sodium hyaluronate.
Background
Hyaluronic Acid (HA), a cosmetic raw material for skin moisturizing, lubrication and repair, is also called hyaluronic acid, is an acidic mucopolysaccharide widely present in the intercellular substance of vertebrate tissues, and HAs a structure formed by repeated and alternate connection of disaccharide units consisting of beta-D-glucuronic acid and beta-D-N-acetylglucosamine, and the molecular weight can range from several thousand to several million daltons. HA was first isolated from bovine vitreous humor in 1934 by American scientists Meyer et al, and HAs been a hot spot in the field of biomedical materials because of its excellent lubricity, moisture retention and viscoelasticity, and excellent biocompatibility and water retention. Usually, Sodium Hyaluronate (SH) is used in its Sodium salt form.
Electrospinning has become an important biomaterial processing technology, and with the development of the technology, the electrospun material is composed of original single components to the existing multi-component, and the structure of the material is diversified, so that conditions are created for the application of the electrospun material in regenerative medicine. The material has good application potential in the field of guiding tissue regeneration through regulating and controlling the components and the structure of the material. By introducing sodium hyaluronate into the electrospinning membrane material, the blood wettability of the membrane can be accelerated, and the wound surface is convenient to adhere and guided tissue regeneration is promoted.
At present, no definite sample pretreatment method exists for the content determination of the sodium hyaluronate in the electrospinning membrane material, so that a sample pretreatment method for the content determination of the sodium hyaluronate in the electrospinning membrane material is expected to be established.
Disclosure of Invention
The invention aims to provide a sample pretreatment method for measuring the content of sodium hyaluronate in an electrospinning membrane material. The invention establishes a rapid, simple and effective sample pretreatment method so as to accurately determine the content of the sodium hyaluronate in the electrospinning membrane material. The method has important significance for research and development, quality control and even clinical application of the products.
The invention provides a sample pretreatment method for measuring the content of sodium hyaluronate in an electrospinning membrane material, which comprises the following steps: and (3) ultrasonically leaching the electrospinning membrane material containing sodium hyaluronate by using water as a leaching medium, collecting the leaching solution, and measuring the content of the sodium hyaluronate in the leaching solution.
In the above method, the leaching ratio of the leaching may be: 1cm2～6cm2The electro-spinning membrane material is leached by 1ml of water;
the frequency of the ultrasonic wave can be 25 KHz-130 KHz;
the ultrasonic leaching time can be 5min-20 min;
the ultrasonic leaching is carried out at room temperature.
The application of ultrasonic water leaching in sample pretreatment for measuring the content of sodium hyaluronate in the electrospinning membrane material also belongs to the protection scope of the invention.
In the application, the leaching proportion of the leaching can be as follows: 1cm2～6cm2The electro-spinning membrane material is leached by 1ml of water;
the frequency of the ultrasonic wave can be 25 KHz-130 KHz;
the ultrasonic leaching time can be 5min-20 min;
the ultrasonic leaching is carried out at room temperature.
Because the electrospun membrane is a hydrophobic material and the sodium hyaluronate is a hydrophilic substance, the method screens out a quick, simple and effective sample pretreatment method by taking water as an extraction medium and researching several different extraction modes, extraction time and extraction proportion, so that a large amount of sodium hyaluronate can be dissolved for subsequent detection, and the property of the electrospun membrane material cannot be damaged; then, the content of the sodium hyaluronate in the leaching solution is quantified according to a corresponding detection method.
The invention provides a sample pretreatment method for simply, conveniently, quickly and efficiently leaching hydrophilic substances such as sodium hyaluronate from an electrospun polymer and then measuring the hydrophilic substances; the sodium hyaluronate under the leaching condition has high recovery rate and good leaching effect.
The invention has the following effects:
the establishment of the sample pretreatment method is beneficial to the quality control of water-soluble natural polymer materials in the electrospinning membrane material products, is beneficial to the research and development and clinical application of the products, and has higher application value;
the method has the following advantages:
(1) the operation is simple, no complex instrument is needed, and the material is convenient to obtain;
(2) the sodium hyaluronate is safely and non-toxic by water leaching, the sodium hyaluronate can be completely eluted, and other interfering substances are prevented from being introduced by proper extraction process and process parameters (such as leaching proportion, temperature and time), so that the accuracy of experimental results is improved;
(3) the result is objective and reliable, and the accuracy is high.
Drawings
FIG. 1 is a D-Glucuronic Acid (GA) standard curve in the sodium hyaluronate assay of example 1 of the present invention.
FIG. 2 is a D-Glucuronic Acid (GA) standard curve in the sodium hyaluronate assay of example 2 of the present invention.
FIG. 3 is a D-Glucuronic Acid (GA) standard curve in the sodium hyaluronate assay of example 3 of the present invention.
FIG. 4 is a D-Glucuronic Acid (GA) standard curve in the sodium hyaluronate assay of comparative example 1 of the present invention.
FIG. 5 is a D-Glucuronic Acid (GA) standard curve in the sodium hyaluronate assay of comparative example 2 of the present invention.
Detailed Description
The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.
Example 1
1. Preliminary screening of leaching pattern
Collecting electrospun membrane (containing sodium hyaluronate) 4cm × 3cm, dividing into four parts, each part is 1cm × 3cm, and extracting at a ratio of 6cm2Perml (calculated according to surface area), adding 1ml of purified water in each part, and respectively processing according to the following 4 ways:
(1) mixing for 2min by using a vortex mixer, and taking out all leaching liquor to a No. I centrifuge tube;
(2) placing the mixture in a shaking table, shaking for 2 hours at 37 ℃, and taking out all leaching liquor to a No. two centrifuge tube;
(3) placing the mixture in a shaking table, shaking for 4 hours at 37 ℃, and taking out all leaching liquor to a No. three centrifugal tube;
(4) placing the mixture in a shaking table, shaking for 6 hours at 37 ℃, and taking out all leaching liquor to a No. four centrifugal tube;
the content of the sodium hyaluronate in the leaching solution is determined according to the method in YY/T1571-2017 tissue engineering medical instrument product sodium hyaluronate. The determination result shows that the detection content exceeds the standard curve range, so the quantitative value of the detection result is inaccurate, but the trend of the detection result can be used as reference, a large amount of sodium hyaluronate can be extracted by processing the membrane product on a vortex mixer for 2min after adding water, and compared with the long-time processing of a shaking table for 6 hours, the vortex extraction efficiency is higher. Therefore, the leaching mode of the shaking table is eliminated, and the further determination of the leaching method of the membrane product through a vortex mixer is considered. The results are shown in FIG. 1 and Table 1.
TABLE 1
Example 2
Research on leaching modes and leaching proportions:
18 pieces of electrospun membrane (without sodium hyaluronate) were taken 2cm × 3cm, 20 μ l of sodium hyaluronate solution (51.1mg/ml) was added to each piece, and freeze-dried for use. The treatment is carried out according to the following 4 ways respectively:
TABLE 2
Test tube 1: taking 0.1ml of the extract from the first step, and adding 0.4ml of water (diluting by 5 times);
test tube 2: 0.25ml of water is added into 0.25ml of water (diluted by 2 times);
test tube 3: taking 0.5ml from the third step;
test tube 4: taking 0.1ml of the extract from the fourth step, and adding 0.4ml of water (diluted by 5 times);
test tube 5: taking 0.25ml from the fifth step, adding 0.25ml of water (diluting by 2 times);
test tube 6: taking 0.5 ml;
the content of the sodium hyaluronate in the leaching solution is determined according to the method in YY/T1571-2017 tissue engineering medical instrument product sodium hyaluronate. The measurement result shows that the ultrasonic treatment is better and more stable than the vortex effect, and the ultrasonic treatment is determined as a leaching mode; the leaching ratio is 1cm2/ml～6cm2The recovery rate is more than 90 percent when the solution is subjected to ultrasound for 5min, the recovery rate meets 100 percent +/-10 percent, and the visible leaching ratio is 1cm2/ml～6cm2Can be used in a dosage of per ml. The results are shown in FIG. 2 and Table 3.
TABLE 3
Example 3
Study of leaching time:
taking 9 pieces of electrospun membrane (without sodium hyaluronate) 3cm × 4cm, adding 40 μ l of sodium hyaluronate solution (51.1mg/ml) in each piece, and freeze-drying for later use. The treatment is carried out according to the following 4 ways respectively:
TABLE 4
Test tube 1: taking 0.1ml of the extract from the first step, and adding 0.4ml of water (diluting by 5 times);
test tube 2: 0.25ml of water is added into 0.25ml of water (diluted by 2 times);
test tube 3: taking 0.5ml from the third step;
the content of the sodium hyaluronate in the leaching solution is determined according to the method in YY/T1571-2017 tissue engineering medical instrument product sodium hyaluronate. The measurement result shows that the leaching ratio is 1cm2/ml～6cm2The recovery rate of the product meets 100% +/-10% when the product is subjected to ultrasonic treatment for 20min, and the visible leaching ratio is 1cm2/ml～6cm2The volume can be changed from 5min to 20min through ultrasonic treatment. The results are shown in FIG. 3 and Table 5.
TABLE 5
Example 4
And (3) detecting the content of sodium hyaluronate in the electrospun membrane:
taking 9 sheets of electrospun membranes (not containing sodium hyaluronate) 2cm × 2.5cm, adding 20 μ l of sodium hyaluronate solution (40mg/ml) into each sheet, and dividing into group 1, group 2 and group 3 according to 3 sheets; another 9 electrospun membranes (without sodium hyaluronate) were taken 2cm × 2.5cm, and 16 μ l of sodium hyaluronate solution (40mg/ml) was added to each membrane, and the membranes were divided into groups 4, 5, and 6 according to 3 tablets. Samples of groups 1-6 were freeze dried for use. Wherein each group 1-3 has a leaching ratio of 1cm2Adding 30ml of purified water into the solution per ml, performing ultrasonic treatment for 5min (ultrasonic frequency is 40KHz), and detecting 0.5ml of leaching solution in each group; groups 4-6 each with a leaching ratio of 6cm2Adding purified water 5ml per ml, performing ultrasonic treatment for 20min (ultrasonic frequency 40KHz), and detecting 0.5ml of leaching solution in each group. The content of the sodium hyaluronate in the leaching solution is determined according to the method in YY/T1571-2017 tissue engineering medical instrument product sodium hyaluronate. The results are shown in Table 6.
TABLE 6
Sodium hyaluronate in the above examples: the weight average molecular weight is 10-100 ten thousand
In the above embodiments, the preparation method of the electrospun membrane (containing sodium hyaluronate) refers to a chinese patent "an antibacterial cation modified absorbable dura mater repair material and its preparation method and application" (patent No. CN106668954A), and the specific preparation method is as follows:
firstly, filling the prepared electrostatic spinning solution into an electrostatic spinning solution supply device; regulating and controlling the supply flow of the electrospinning solution to be 0.1-20 ml/h, specifically 1 ml/h through a flow pump; adjusting the distance between the high-pressure generating end and the collecting device to be 3-30 cm, specifically 15 cm; the high voltage generator provides high voltage for the electrostatic spinning process, and the voltage can be regulated and controlled between 0.1 kilovolt and 40 kilovolt (specifically 15 kilovolt); connecting a roller with the rotation speed of 100-; collecting on a rotating roller to obtain the electrospun membrane. And (3) coating the sodium hyaluronate solution on the surface of the electrospinning membrane in a casting manner to obtain the electrospinning membrane coated with the natural polymer solution on the surface, and freeze-drying to obtain the composite membrane containing the three-dimensional porous polymer layer.
The preparation method of the blank electrospun membrane (without sodium hyaluronate) is different from the preparation method of the electrospun membrane in that the electrospun membrane collected on the rotary drum is directly frozen and dried without being coated with a sodium hyaluronate solution.
D-Glucuronic Acid (GA) used in the present invention: biometrics bioengineering (Shanghai) Inc., A600482-0005; sodium tetraborate decahydrate (Na2B4O7 · 10H 2O): biotechnology engineering (Shanghai) GmbH, A610480-0500; carbazole: chemical agents of the national drug group, ltd, 80030280; ultrasonic cleaning machines (ultrasonic frequency 25KHz, 40KHz, 130 KHz); ultraviolet spectrophotometer, UV-2450.
As can be seen from example 1: by comparing the two leaching modes of the shaking table and the vortex, the leaching mode of the shaking table is eliminated, and the further determination of the leaching method of the membrane product through the vortex mixer is considered.
As can be seen from example 2: by comparing the ultrasonic leaching mode and the vortex leaching mode, the ultrasonic treatment is found to be better and more stable than the vortex effect, so the vortex mode is eliminated, the ultrasonic treatment is determined as the optimal leaching mode, and in combination with example 3, the leaching ratio of the sample pretreatment is found to be 1cm2/ml-6cm2The recovery rate can be 100% +/-10% in both the case of/ml and the case of 5-20 min of ultrasound.
As can be seen from example 4: the average recovery rate of the sodium hyaluronate of the electrospun membrane containing the known content of the sodium hyaluronate is 101.72%, and the precision is 0.06%, which shows that the method is relatively stable and is suitable for being used as a conventional method for detecting the content of the sodium hyaluronate in the electrospun membrane material.
The ultrasonic treatment mode can simply and efficiently leach the sodium hyaluronate from the electrospun membrane, and the method can meet the requirements for the frequency of a common ultrasonic cleaning machine, greatly improves the timeliness and the accuracy of an experiment, and has guiding significance for the content and the clinical use of the water-soluble active ingredients such as the sodium hyaluronate on the electrospun membrane.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Comparative example 1
Study of leaching time:
12 pieces of electrospun membrane (without sodium hyaluronate) 2cm × 2.5cm were taken, 20 μ l of sodium hyaluronate solution (40mg/ml) was added to each piece, and freeze-dried for later use. The method comprises the following four ways of processing, and under the condition that other conditions are not changed, the recovery rate of the sodium hyaluronate with the leaching time of more than 20min (taking 40min as a research object) and the leaching time of less than 5min (taking 1min as a research object) is examined by using 40KHz ultrasonic frequency:
TABLE 7
Test tube 1: taking 0.1ml of the extract from the first step, and adding 0.4ml of water (diluting by 5 times);
test tube 2: 0.5ml is taken out;
test tube 3: taking 0.1ml from the third step, adding 0.4ml of water (diluted by 5 times);
test tube 4: taking 0.5ml from the fourth step;
the content of the sodium hyaluronate in the leaching solution is determined according to the method in YY/T1571-2017 tissue engineering medical instrument product sodium hyaluronate. The measurement result shows that when the leaching ratio is 1cm2/ml～6cm2The recovery rate of sodium hyaluronate decreased at/ml, 40min and 1min of ultrasound, considering that the leaching time was too short to allow sufficient leaching and the leaching time was too long to allow the electrospun membrane to be damaged to generate other interfering components. Therefore, from the aspects of detection accuracy and detection time period, the extraction time is preferably 5min to 20 min. The results are shown in FIG. 4 and Table 8.
TABLE 8
Comparative example 2
Study of leaching ratio:
12 pieces of electrospun membrane (without sodium hyaluronate) 2cm × 2.5cm were taken, 20 μ l of sodium hyaluronate solution (40mg/ml) was added to each piece, and freeze-dried for later use. Respectively treating in four ways, and observing the leaching ratio of more than 6cm with 40KHz ultrasonic frequency under the condition of unchanged other conditions2Per ml (in 8 cm)2The/ml is the object of study) and the leaching ratio is less than 1cm2Per ml (at 0.5 cm)2Per ml subject) recovery of sodium hyaluronate:
TABLE 9
Test tube 1: taking 0.1ml of the extract from the first step, and adding 0.4ml of water (diluting by 5 times);
test tube 2: 0.5ml is taken out;
test tube 3: taking 0.1ml from the third step, adding 0.4ml of water (diluted by 5 times);
test tube 4: taking 0.5ml from the fourth step;
the content of the sodium hyaluronate in the leaching solution is determined according to the method in YY/T1571-2017 tissue engineering medical instrument product sodium hyaluronate. The measurement result shows that when the leaching time is 5min-20min, the leaching proportion is 8cm2At/ml, the recovery rate of the sodium hyaluronate is lower than 90%, after the leaching proportion is increased, the leaching ratio means that less water is used for leaching a larger area of sample, partial electrospun membrane sample is not completely immersed in the water, so that the completeness of leaching is influenced, and the leaching concentration is increased due to the reduction of leaching media and exceeds the upper limit of the standard curve concentration in the method, so that the quantification is inaccurate; when the leaching time is 5min to 20min and the leaching ratio is 0.5cm2At the time of per ml, although the recovery rate of the sodium hyaluronate is more than 90%, the leaching is considered to be complete, on one hand, unnecessary waste is caused due to the increase of the volume of the leaching medium water, on the other hand, the leaching concentration is reduced due to the increase of the volume of the water, and the concentration is preferably close to the lower limit of the concentration of the standard curve in the method, and the detection concentration is preferably positioned at the middle concentration of the standard curve, so that the result quantification is more accurate. Therefore, the extraction ratio is recommended to be 1cm in combination with the standard curve concentration range and the water saving angle in the method2/ml～6cm2Preferably,/ml. The results are shown in FIG. 5 and Table 10.
Watch 10
Claims (8)
1. A sample pretreatment method for measuring the content of sodium hyaluronate in an electrospinning membrane material comprises the following steps: and (3) ultrasonically leaching the electrospinning membrane material containing sodium hyaluronate by using water as a leaching medium, collecting the leaching solution, and measuring the content of the sodium hyaluronate in the leaching solution.
2. The method of claim 1, wherein: said leachedThe leaching proportion is as follows: 1cm2～6cm2The electrospun membrane material was leached with 1ml of water.
3. The method according to claim 1 or 2, characterized in that: the ultrasonic frequency of the ultrasonic leaching is 25 KHz-130 KHz.
4. The method according to any one of claims 1-3, wherein: the ultrasonic leaching time is 5min to 20 min;
the ultrasonic leaching is carried out at room temperature.
5. The application of ultrasonic water leaching in sample pretreatment for measuring the content of sodium hyaluronate in an electrospinning membrane material.
6. Use according to claim 5, characterized in that: the leaching proportion of leaching is as follows: 1cm2～6cm2The electrospun membrane material was leached with 1ml of water.
7. Use according to claim 5, characterized in that: the ultrasonic frequency of the ultrasonic leaching is 25 KHz-130 KHz.
8. Use according to claim 5, characterized in that: the ultrasonic leaching time is 5min to 20 min;
the ultrasonic leaching is carried out at room temperature.
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